Three-dimensional (3D) tissue models have significantly improved our understanding of structure/function relationships and promise to lead to new advances in regenerative medicine. However, despite the expanding diversity of 3D tissue fabrication methods, in vitro approaches for functional assessments have been relatively limited. Herein, we describe the guidance of primary endothelial cells (ECs) in an agarose hydrogel scaffold that is chemically patterned with an immobilized concentration gradient of vascular endothelial growth factor 165 (VEGF165) using multiphoton laser patterning of VEGF165. This is the first demonstration of this patterning technology to immobilize proteins; and the first demonstration of immobilized VEGF165 to guide endothelial cell growth and differentiation in 3D environments. It is particularly compelling that this 3D hydrogels provide an excellent biomimetic environment for stem cell niche, thereby offering a new approach to study stem cell biology. In this thesis, we focused on the retinal stem cell niche, investigating cellular interactions between retinal stem and progenitor cells (RSPCs) and endothelial cells (ECs). By using this 3D in vitro model, we demonstrated the synergistic interactions between RSPCs and ECs wherein RSPCs migrated into 3D gels only in the presence of ECs and RSPCs stabilized EC tubular-like formations. Moreover, we characterized the contact-mediated effects of ECs on RSPC fate in terms of proliferation and differentiation.

Three-dimensional (3D) tissue models have significantly improved our understanding of structure/function relationships and promise to lead to new advances in regenerative medicine. However, despite the expanding diversity of 3D tissue fabrication methods, in vitro approaches for functional assessments have been relatively limited. Herein, we describe the guidance of primary endothelial cells (ECs) in an agarose hydrogel scaffold that is chemically patterned with an immobilized concentration gradient of vascular endothelial growth factor 165 (VEGF165) using multiphoton laser patterning of VEGF165. This is the first demonstration of this patterning technology to immobilize proteins; and the first demonstration of immobilized VEGF165 to guide endothelial cell growth and differentiation in 3D environments. It is particularly compelling that this 3D hydrogels provide an excellent biomimetic environment for stem cell niche, thereby offering a new approach to study stem cell biology. In this thesis, we focused on the retinal stem cell niche, investigating cellular interactions between retinal stem and progenitor cells (RSPCs) and endothelial cells (ECs). By using this 3D in vitro model, we demonstrated the synergistic interactions between RSPCs and ECs wherein RSPCs migrated into 3D gels only in the presence of ECs and RSPCs stabilized EC tubular-like formations. Moreover, we characterized the contact-mediated effects of ECs on RSPC fate in terms of proliferation and differentiation.